Antisense oligonucleotides (ASOs) are emerging as a promising class of therapeutics for neurological diseases. When injected directly into cerebrospinal fluid, ASOs distribute broadly across brain regions and exert long-lasting therapeutic effects. However, many phosphorothioate (PS)-modified gapmer ASOs show transient motor phenotypes when injected into the cerebrospinal fluid, ranging from reduced motor activity to ataxia or acute seizure-like phenotypes. Using a behavioral scoring assay customized to reflect the timing and nature of these effects, we show that both sugar and phosphate modifications influence acute motor phenotypes. Among sugar analogs, DNA induces the strongest motor phenotypes while 2′-substituted RNA modifications improve the tolerability of PS ASOs. Reducing the PS content of gapmer ASOs, which contain a stretch of PS-DNA, improves their toxicity profile, but in some cases also reduces efficacy or duration of effect. We show that this acute toxicity is not mediated by major nucleic acid sensing immune pathways. Formulating ASOs with divalent ions before injection and avoiding phosphate-based buffers modestly improved tolerability through mechanisms at least partially distinct from reduced PS content. Overall, our work identifies and quantifies an understudied aspect of oligonucleotide toxicology in the CNS, explores its mechanism, and presents platform-level medicinal chemistry and formulation approaches that improve tolerability of this class of compounds.

中文翻译：

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量化和减轻中枢神经系统中反义寡核苷酸诱导的运动表型


反义寡核苷酸 （ASO） 正在成为一类很有前途的神经系统疾病治疗方法。当直接注射到脑脊液中时，ASO 广泛分布在大脑区域并发挥持久的治疗效果。然而，许多硫代磷酸酯 （PS） 修饰的间隙体 ASO 在注射到脑脊液时表现出短暂的运动表型，范围从运动活动减少到共济失调或急性癫痫样表型。使用为反映这些影响的时间和性质而定制的行为评分分析，我们表明糖和磷酸盐修饰都会影响急性运动表型。在糖类似物中，DNA 诱导最强的运动表型，而 2' 取代的 RNA 修饰提高了 PS ASO 的耐受性。降低含有一段 PS-DNA 的间隙 ASO 的 PS 含量可以改善其毒性特征，但在某些情况下也会降低疗效或效果持续时间。我们表明这种急性毒性不是由主要核酸感应免疫途径介导的。在注射前用二价离子配制 ASO 并避免使用磷酸盐缓冲液，通过至少部分不同于降低的 PS 含量的机制，适度提高了耐受性。总体而言，我们的工作确定并量化了 CNS 中寡核苷酸毒理学研究不足的方面，探索了其机制，并提出了提高此类化合物耐受性的平台级药物化学和制剂方法。